Multi-index test strip and application thereof

Abstract

The invention provides a multi-index test strip which sequentially comprises a sample pad, a combination pad, a reaction film and an absorption pad in the flowing direction of a sample, the combination pad comprises a first fluorescent microsphere-labeled antibody and a second fluorescent microsphere-labeled antibody, the first fluorescent microsphere-labeled antibody is combined with a first antigen, and the second fluorescent microsphere-labeled antibody is combined with a second antigen; a detection line and a quality control line are sequentially arranged on the reaction film in the sample flowing direction, and the detection line comprises a first capture antibody paired with the antibody marked by the first fluorescent microsphere and a second capture antibody paired with the antibody marked by the second fluorescent microsphere; wherein the first antigen in the sample to be detected is more difficult to detect than the second antigen, and the particle size of the first fluorescent microspheres is smaller than that of the second fluorescent microspheres. According to the multi-index test strip, the sensitivity and specificity during multi-index detection can be improved, mutual interference during multi-index detection can be reduced, the linear range and correlation can be improved, and the detection cost cannot be remarkably increased.

Description

Technical Field

[0001] The present application belongs to the field of pathogen protein marker detection. Specifically, the present application relates to a multi-indicator test strip and its use. Background Art

[0002] Lateral flow immunoassay (LFIA) is a common technology based on immune platform for rapid detection of antigens, antibodies or small molecules to be tested. The typical application of this technology is test strips, and its structure is mainly divided into four parts: sample pad, conjugate pad, reaction membrane and absorption pad. The conjugate pad is fixed with antigens or antibodies labeled with tracers, and the reaction membrane is coated with antigens or antibodies for capture. During the test, the sample to be tested is dripped onto the sample pad, and the analyte in the sample will be chromatographed along the direction of the reaction membrane by capillary force, and first bind to the antigen or antibody fixed on the conjugate pad, and then be captured by the antigen or antibody coated on the reaction membrane, thereby forming a detectable signal at a fixed position on the reaction membrane. Then, the corresponding signal is interpreted by the naked eye or instrument, and the content of the analyte in the sample can be judged.

[0003] Lateral flow immunoassay is also called lateral flow immunochromatography. Common lateral flow immunoassays are mainly divided into three types according to the different tracers: colloidal gold immunochromatography, latex immunochromatography and fluorescent immunochromatography. Among them, colloidal gold immunochromatography generally uses gold particles with a particle size of 1 to 100 nm as tracers, and labels antigens or antibodies through electrostatic adsorption. The signal is a colored strip, and the result is interpreted by visual inspection or instrument detection of grayscale value; latex immunochromatography generally uses latex microspheres with a particle size of 100 to 500 nm as tracers, and labels antigens or antibodies through covalent bonding. The signal is a colored strip, and the result is also interpreted by visual inspection or instrument detection of grayscale value; fluorescent immunochromatography generally uses fluorescent microspheres with a particle size of 100 to 500 nm or quantum dots with a particle size of 2-20 nm as tracers, and labels antigens or antibodies through covalent bonding. The signal is a fluorescent strip with fixed excitation and emission wavelengths, and the result is interpreted by instrument detection of fluorescence intensity.

[0004] When multiple indicators need to be tested on a test strip at the same time, there may be interference between the indicators, affecting the sensitivity and specificity of a single indicator. To address this problem, some studies have used latex microspheres of different colors to distinguish different indicators, but this operation has the disadvantage that mixed colors are not easy to distinguish; some studies have used fluorescent microspheres or quantum dots with different emission wavelengths to distinguish different indicators, but in fluorescent immunochromatography, the types of filters in the equipment will be increased, increasing the detection cost. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present application is to provide a multi-indicator test strip and its use, which can improve the sensitivity of multi-indicator detection and reduce non-specific interference without significantly increasing the detection cost.

[0006] Specifically, this application involves the following contents:

[0007] 1. A multi-index test strip, which comprises a sample pad, a binding pad, a reaction membrane and an absorption pad in sequence along the sample flow direction;

[0008] The conjugate pad comprises an antibody labeled with a first fluorescent microsphere and an antibody labeled with a second fluorescent microsphere, wherein the antibody labeled with the first fluorescent microsphere binds to a first antigen, and the antibody labeled with the second fluorescent microsphere binds to a second antigen;

[0009] A detection line and a quality control line are sequentially arranged on the reaction membrane along the sample flow direction, wherein the detection line comprises a first capture antibody paired with the antibody labeled with the first fluorescent microsphere, and a second capture antibody paired with the antibody labeled with the second fluorescent microsphere;

[0010] The first antigen in the sample to be tested is more difficult to detect than the second antigen, and the particle size of the first fluorescent microsphere is smaller than the particle size of the second fluorescent microsphere.

[0011] 2. The multi-indicator test strip according to item 1, wherein the first capture antibody is closer to the absorption pad than the second capture antibody.

[0012] 3. The multi-index test strip according to item 2, wherein the distance between the first capture antibody and the second capture antibody is 1 mm to 10 mm.

[0013] 4. The multi-index test strip according to any one of items 1 to 3, wherein the particle size of the first fluorescent microsphere is 50 nm to 400 nm.

[0014] 5. The multi-index test strip according to any one of items 1 to 4, wherein the particle size of the second fluorescent microsphere is 200 nm to 1000 nm.

[0015] 6. The multi-index test strip according to any one of items 1 to 5, wherein the particle size of the second fluorescent microsphere is larger than that of the first fluorescent microsphere by more than 50 nm, preferably more than 100 nm.

[0016] 7. The multi-indicator test strip according to any one of items 1 to 6, wherein the first antigen in the test sample is more difficult to detect than the second antigen, comprising:

[0017] The first antigen has a lower concentration than the second antigen;

[0018] The first antigen has a lower detection sensitivity than the second antigen;

[0019] The first antigen is more susceptible to interference than the second antigen. 8. The multi-indicator test strip according to any one of items 1 to 7, wherein the first antigen is placental growth factor (PlGF) and the second antigen is soluble fms-like tyrosine kinase-1 (sFlt-1).

[0020] 9. According to the multi-indicator test strip according to any one of items 1 to 7, the first antigen is pepsinogen II (PGII) and the second antigen is pepsinogen I (PGI).

[0021] 10. A multi-indicator detection card, comprising a card housing and a multi-indicator test strip according to any one of items 1 to 9 in the card housing.

[0022] 11. Use of the multi-index test strip described in any one of items 1 to 9 in the preparation of a product for detecting eclampsia.

[0023] 12. Use of the multi-index test strip described in any one of items 1 to 9 in the preparation of a product for detecting gastric function.

[0024] Compared with the prior art, the multi-index test strips and multi-index test cards of the present application have achieved the following technical effects:

[0025] 1. It can improve the sensitivity and specificity of multi-index detection.

[0026] 2. It can reduce mutual interference during multi-index detection and improve linear range and correlation.

[0027] 3 Because the costs of fluorescent microspheres of different particle sizes are relatively similar and there is no change to the detection equipment, this application will not significantly increase the detection cost.

[0028] The multi-index test strips and multi-index test cards of this application can be used in major hospital departments, as well as in grassroots community hospitals and even families. At the same time, this technology can be combined with clinical and other laboratory indicators and promoted to different application fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the structure of the multi-indicator test strip of this application.

[0030] Reference numerals

[0031] 1 sample pad, 2 binding pad, 3 reaction membrane, 4 absorption pad, 11 sample to be tested, 12 first antigen, 13 second antigen, 21 first fluorescent microsphere labeled antibody, 22 second fluorescent microsphere labeled antibody, 31 second capture antibody; 32 first capture antibody, 33 quality control line corresponding antibody. DETAILED DESCRIPTION

[0032] The present application is further described below in conjunction with examples. It should be understood that the examples are only used to further describe and illustrate the present application and are not used to limit the present application.

[0033] Unless otherwise defined, the technical and scientific terms in this specification have the same meaning as those generally understood by those skilled in the art. Although similar or identical methods and materials as described herein may be used in experiments or practical applications, materials and methods are described herein below. In the event of a conflict, the present specification including the definitions therein shall prevail. In addition, materials, methods and examples are provided for illustration only and are not restrictive. The present application is further described below in conjunction with specific embodiments, but is not intended to limit the scope of the present application.

[0034] The present application provides a multi-indicator test strip. The structure of the test strip is as follows Figure 1 As shown, the multi-index test strip includes a sample pad 1, a binding pad 2, a reaction membrane 3 and an absorption pad 4 in sequence along the sample flow direction.

[0035] The sample pad 1 is made of glass cellulose membrane and is used for dropping test samples.

[0036] The material of the conjugate pad 2 is glass cellulose membrane. The conjugate pad 2 contains antibodies labeled with fluorescent microspheres of different particle sizes, specifically, it can contain antibodies 21 labeled with a first fluorescent microsphere and antibodies 22 labeled with a second fluorescent microsphere, the first fluorescent microsphere labeled antibody 21 binds to the first antigen 12, and the second fluorescent microsphere labeled antibody 22 binds to the second antigen 13. The first antigen 12 is more difficult to detect than the second antigen 13 in the sample to be tested, and the particle size of the first fluorescent microsphere is smaller than the particle size of the second fluorescent microsphere.

[0037] The first antigen 12 in the test sample is more difficult to detect than the second antigen 13, which refers to various more difficult detection situations known to those skilled in the art, for example, the first antigen 12 may be lower than the second antigen concentration 13, and the lower may be 2 times lower, 5 times lower, 10 times lower, 20 times lower, lower, 30 times lower, 40 times lower, 50 times lower, 60 times lower, 70 times lower, 80 times lower, 90 times lower, 100 times lower, 200 times lower, 500 times lower, or even 1000 times lower or more. For example, the two indicators related to eclampsia, the content of PlGF in serum is several times or even hundreds of times lower than that of another indicator sFlt-1. The level of sFlt-1 in the serum of normal pregnant women at different gestational weeks is generally in the range of 500 to 10000 pg / mL, and the level of PlGF in serum is generally in the range of 20 to 1000 pg / mL. In the serum of pregnant women with preeclampsia or eclampsia, the level of PlGF is lower and the level of sFlt-1 is higher.

[0038] The first antigen 12 is more difficult to detect than the second antigen 13, or the first antigen 12 has a lower detection sensitivity than the second antigen 13, and lower can be 2 times lower, 5 times lower, 10 times lower, 20 times lower, lower, 30 times lower, 40 times lower, 50 times lower, 60 times lower, 70 times lower, 80 times lower, 90 times lower, 100 times lower, 200 times lower, 500 times lower, or even 1000 times lower or more. For example, two indicators related to inflammation are conventional C-reactive protein (CRP) and high-sensitivity C-reactive protein (hs-CRP), the normal reference value of CRP is <10mg / L, and the normal reference value of hs-CRP is <1mg / L, and the detection sensitivity of hs-CRP is lower.

[0039] The first antigen 12 may be more difficult to detect than the second antigen 13, or the first antigen 12 may be more susceptible to interference than the second antigen 13. For example, the two indicators related to gastric function, pepsinogen I (PGI) and pepsinogen II (PGII), have certain homology and may interfere with each other to a certain extent. The normal reference value of PGI is about 70-200 ng / mL, and the normal reference value of PGII is about 0-15 ng / mL. After preliminary testing, 50 ng / mL of PGII has no interference with PGI, and 200 ng / mL of PGI has no interference with PGII, that is, PGII with 3 times the upper limit of the normal value has no interference with PGI, and PGI with 1 times the upper limit of the normal value has no interference with PGII, indicating that PGII is more susceptible to interference than PGI.

[0040] The type of fluorescent microspheres can be any fluorescent microspheres known in the art that are suitable for fluorescent immunochromatography, for example, ordinary polystyrene fluorescent microspheres with fluorescent substances, time-resolved fluorescent microspheres doped with rare earth elements, and the like.

[0041] The particle size of the first fluorescent microsphere and the particle size of the second fluorescent microsphere are not limited, as long as the particle size of the first fluorescent microsphere is smaller than the particle size of the second fluorescent microsphere and is suitable for fluorescent immunochromatography.

[0042] In a specific embodiment, the particle size of the first fluorescent microsphere is 50nm to 400nm, for example, it can be 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, and any range between these values.

[0043] In a specific embodiment, the particle size of the second fluorescent microsphere is 200nm to 1000nm, for example, it can be 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, 500nm, 510nm, 520nm, 530nm, 540nm, 550nm, 560nm, 570nm, 580nm 0nm, 80nm, 810nm, 820nm, 830nm, 840nm, 850nm, 860nm, 870nm, 880nm, 890nm, 900nm, 910nm, 920nm, 930nm, 940nm, 950nm, 960nm, 970nm, 980nm, 990nm, 1000nm, and any range between these values.

[0044] In a specific embodiment, the particle size of the first fluorescent microsphere is 100-200 nm, and the particle size of the second fluorescent microsphere is 200-300 nm.

[0045] In a specific embodiment, the particle size of the second fluorescent microsphere is larger than that of the first fluorescent microsphere by more than 50 nm, for example, it can be 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 50nm, 260nm, 265nm, 270nm, 275nm, 280nm, 285nm, 290nm, 295nm, 300nm, and any range between these values.

[0046] In a specific embodiment, the particle size of the second fluorescent microsphere is greater than that of the first fluorescent microsphere by more than 100 nm.

[0047] In the multi-indicator test strip of the present application, the first antigen is more difficult to detect than the second antigen, and the particle size of the first fluorescent microsphere is smaller than that of the second fluorescent microsphere. Because the particle size of the fluorescent microsphere is small, the chromatography speed is fast, and the first fluorescent microsphere is first bound by the corresponding first capture antibody. At this time, after the second antigen is bound to the labeled antibody, because the particle size of the second fluorescent microsphere is large, the chromatography speed is slow, and it has not reached the position of the first capture antibody, so it will not interfere with the binding of the first antigen, and can also improve the capture efficiency of the first antigen.

[0048] The reaction membrane 3 is made of nitrocellulose membrane. A detection line and a quality control line 32 are sequentially arranged on the reaction membrane 3 along the sample flow direction. The detection line comprises a first capture antibody 32 paired with the antibody labeled with the first fluorescent microsphere, and a second capture antibody 31 paired with the antibody labeled with the second fluorescent microsphere. The number of detection lines can be 2.

[0049] In a specific embodiment, the first capture antibody 32 is closer to the absorption pad 4 than the second capture antibody 31 .

[0050] The capture antibody of the second antigen is located close to the binding pad, and the overall reaction (chromatography) time is short, resulting in an insufficient reaction compared to the capture antibody of the first antigen. Using fluorescent microspheres with large particle sizes to label the detection antibody of the second antigen can reduce the chromatography speed of the antigen-labeled antibody complex of the second antigen, prolong the reaction time, compensate for the insufficient reaction caused by the close position, and improve the detection sensitivity of the second antigen.

[0051] In a specific embodiment, the distance between the first capture antibody 32 and the second capture antibody 31 is 1 to 10 mm, for example, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, and any range therebetween.

[0052] In a specific embodiment, there are two detection lines, and the two detection lines contain the first capture antibody 32 and the second capture antibody 31 respectively.

[0053] The quality control line 32 is formed by coating a solution containing goat anti-mouse IgG polyclonal antibody.

[0054] The content of each antibody labeled with microspheres of different particle sizes on the conjugate pad 2 and each capture antibody on the reaction membrane 3 can be adjusted according to actual needs.

[0055] In a specific embodiment, the content of each antibody labeled with microspheres of different particle sizes on the conjugate pad 2 is 0.1-10 μg / cm 2 .

[0056] In a specific embodiment, the content of each capture antibody on the reaction membrane 3 is 0.2-1 μg / cm 2 .

[0057] In a specific embodiment, the first antigen 12 is placental growth factor (PlGF), and the second antigen 13 is soluble fms-like tyrosine kinase-1 (sFlt-1). The multi-index test strip of the present application can detect eclampsia. Preeclampsia is a serious pregnancy complication, and the main clinical manifestations are increased blood pressure and proteinuria around 20 weeks of pregnancy, and symptoms such as headache, dizziness, nausea, vomiting, and upper abdominal discomfort may occur. Eclampsia develops from preeclampsia into more serious symptoms, causing convulsions or coma. Many studies have confirmed that placental growth factor and soluble fms-like tyrosine kinase-1 are closely related to the onset of preeclampsia and are important factors in the onset of preeclampsia. The placenta of patients with preeclampsia overexpresses sFlt-1 and removes vascular endothelial growth factor (VEGF) and PlGF, which can cause widespread endothelial dysfunction, leading to multi-organ disease. Under normal circumstances, the PlGF level in the serum of pregnant women increases in the early stages of pregnancy and gradually decreases to full-term pregnancy as the pregnancy progresses; the sFlt-1 level remains stable in the early and mid-pregnancy and steadily increases to full-term pregnancy in the late stages. The serum sFlt-1 level of pregnant women with preeclampsia is higher than that of normal pregnant women, while the PlGF level is lower than that of normal pregnant women. Determining the sFlt-1 / PlGF ratio is more valuable than detecting a single indicator. Therefore, by determining the sFlt-1 / PlGF ratio in the serum of pregnant women and combining it with other clinical examinations, the diagnostic accuracy of preeclampsia can be improved.

[0058] In another specific embodiment, the first antigen 12 is PGII, and the second antigen 13 is PGI. The multi-index test strip of the present application can quantitatively detect PGI / PGII in human serum in vitro, which is used to evaluate the function of gastric acid secreting gland cells and assist in the diagnosis of fundic gland lesions. The PGI level in serum and the PGI / PGII ratio can be used as indicators to judge the degree of fundic gland mucosal atrophy. When PGI < 70 ng / ml and the PGI / PGII ratio < 3.0 are used as cutoff values, the diagnosis rate of fundic gland mucosal atrophy diseases is the highest.

[0059] In a specific embodiment, a multi-index test strip comprises a sample pad 1, a conjugation pad 2, a reaction membrane 3 and an absorption pad 4 in sequence along the sample flow direction; the conjugation pad 2 comprises an antibody 21 labeled with a first fluorescent microsphere and an antibody 22 labeled with a second fluorescent microsphere, the antibody 21 labeled with the first fluorescent microsphere binds to a first antigen 12, and the antibody 22 labeled with the second fluorescent microsphere binds to a second antigen 13; a detection line and a quality control line 32 are sequentially arranged on the reaction membrane 3 along the sample flow direction, the detection line comprises a first capture antibody 32 paired with the antibody labeled with the first fluorescent microsphere, and a second capture antibody 31 paired with the antibody labeled with the second fluorescent microsphere, the first capture antibody 32 is closer to the absorption pad 4 than the second capture antibody 31; wherein the first antigen 12 in the sample to be tested is more difficult to detect than the second antigen 13, and the particle size of the first fluorescent microsphere is smaller than the particle size of the second fluorescent microsphere.

[0060] The present application also provides a multi-indicator test card, which includes a card housing and any of the above-mentioned multi-indicator test strips in the card housing. The specific structure of the card housing can be adjusted according to different actual application scenarios.

[0061] The multi-indicator test strip of the present application can be prepared according to methods known in the art.

[0062] The multi-indicator test strip or multi-indicator test card of the present application can be tested by a fluorescent immunoassay analyzer.

[0063] The test card and test kit prepared in this application can improve the sensitivity and specificity of multi-index detection. Thereby effectively carrying out joint detection, and because the cost of fluorescent microspheres of different particle sizes is relatively close and there is no change to the detection equipment, this application will not significantly increase the detection cost. In addition to being used in major hospital departments, the multi-index test strips and multi-index test cards of this application can also be used in grassroots community hospitals and even families. At the same time, this technology can be combined with clinical and other laboratory indicators and promoted to different application fields.

[0064] The present application also provides the use of any of the above-mentioned multi-indicator test strips in the preparation of a product for detecting eclampsia.

[0065] Example

[0066] In the following examples, unless otherwise specified, the materials used in the examples are all commercially available products. Among them, fluorescent microspheres were purchased from Hangzhou Boyue Biotechnology Co., Ltd., and the product numbers EU0100C2, EU0200C1 and EU0300C1 correspond to microspheres with particle sizes of 100 nm, 200 nm and 300 nm, respectively.

[0067] Example 1 Preparation of Eclampsia Dual Detection Card (Fluorescence Immunochromatography)

[0068] (1) Fluorescent microspheres of different sizes labeled with mouse anti-PlGF and mouse anti-sFlt-1 monoclonal antibodies

[0069] Measure 0.09 mg of mouse anti-PlGF monoclonal antibody for labeling, dilute to 0.18 ml with purified water and mix well. Then add 1.08 ml of purified water and 0.075 ml of 500 mM MES solution, mix well, add 0.15 ml of fluorescent microspheres with a particle size of 200 nm, and mix with a mixer at room temperature and away from light for 15 minutes. Then add 0.015 ml of 10 mg / mL EDC, mix well immediately, and mix with a mixer at room temperature and away from light for 60 minutes. Centrifuge at 12000-13000 rpm and 4°C for 10 minutes. Carefully discard the supernatant, add 1.5 ml of 25 mM MES, and sonicate for 2 seconds * 10 times to re-dissolve (if the precipitate is difficult to re-dissolve, increase the number of sonications until it is completely re-dissolved). Repeat the centrifugation and 25mM MES washing and re-dissolving process once, then centrifuge for the third time at 4℃ for 10 minutes, carefully discard the supernatant, add 1.5ml fluorescent resuspension solution, and sonicate for 2 seconds*10 times to re-dissolve (if the precipitate is difficult to re-dissolve, increase the number of sonications until it is completely re-dissolved), and obtain PlGF antibody labeled with 200nm fluorescent microspheres. Store at 2-8℃ for future use, with a shelf life of 1 month.

[0070] The same method was used to prepare 100nm fluorescent microsphere-labeled PlGF antibody, 300nm fluorescent microsphere-labeled PlGF antibody, 100nm fluorescent microsphere-labeled sFlt-1 antibody, 200nm fluorescent microsphere-labeled sFlt-1 antibody, and 300nm fluorescent microsphere-labeled sFlt-1 antibody.

[0071] (2) Preparation of fluorescent microsphere binding pad

[0072] Before each use, the fluorescent microsphere-labeled PlGF antibody and fluorescent microsphere-labeled sFlt-1 antibody solutions need to be balanced to room temperature and then ultrasonically dissolved for 2 seconds * 10 times. When mixing, surface area correction is performed, that is, both indicators are labeled with 200nm particle size microspheres, and they are diluted 20 times with fluorescent resuspension solution before use (the volume ratio of the two fluorescent labeled antibody solutions to the fluorescent resuspension solution is 0.5:0.5:9). Spray the diluted fluorescent microsphere-labeled antibody on the glass fiber membrane at a spraying volume of 2.5μL / cm 2 After spraying, place it in a drying room to dry for 3-24 hours to obtain the fluorescent conjugate pad, and then store it in a dryer at room temperature. When the particle size is reduced to 100nm, the amount of fluorescent labeled antibody added is 4 times that of 200nm; when the particle size is increased to 300nm, the amount of fluorescent labeled antibody added is 0.5 times that of 200nm.

[0073] The preparation of the fluorescent resuspension is as follows:

[0074] Weigh 6.06 g of tris(hydroxymethyl)aminomethane, 20.00 g of sucrose, 5.00 g of bovine serum albumin, 1.00 g of PEG20000, 1.00 g of Triton X-100, and 1.0 mL of PC300, add 700.0 mL of purified water, stir to fully dissolve, add appropriate amount of concentrated hydrochloric acid, stir evenly, adjust the pH to 7.4, stir to dissolve, and dilute to 1000.0 mL.

[0075] (3) Preparing a test line and a quality control line on a nitrocellulose membrane to obtain a reaction membrane.

[0076] Take 5.0 mg of mouse anti-PlGF monoclonal antibody for coating, add it to 3.0 mL 0.05 M PBS, mix it thoroughly and make it to a final volume of 5.0 mL with 0.05 M PBS buffer, the final concentration of the antibody is 1.0 mg / mL. Take 5.0 mg of mouse anti-sFlt-1 monoclonal antibody for coating, add it to 3.0 mL 0.05 M PBS, mix it thoroughly and make it to a final volume of 5.0 mL with 0.05 M PBS buffer, the final concentration of the antibody is 1.0 mg / mL. Take 5.0 mg of goat anti-mouse IgG polyclonal antibody (from Luoyang Baitai Biotechnology Co., Ltd.), add it to 3.0 mL 0.05 M PBS buffer, mix it thoroughly and make it to a final volume of 5.0 mL with 0.05 M PBS buffer, the final concentration of the goat anti-mouse IgG polyclonal antibody is 1.0 mg / mL.

[0077] The solution containing mouse anti-PlGF, sFlt-1 monoclonal antibody and goat anti-mouse IgG for coating was sprayed at a rate of 10 μL / cm 2 Spray onto the nitrocellulose membrane to form 2 test lines and 1 quality control line, including the following along the sample chromatography direction (i.e., from the binding pad to the absorbent paper): test line T1 (sFlt-1), test line T2 (PlGF) and quality control line C (sheep anti-mouse IgG). Put the reaction membrane coated with the test line and quality control line into the drying workshop for 3-72 hours.

[0078] (4) Paste the sample pad, fluorescent microsphere binding pad, reaction membrane, and absorption pad on the PVC board, so that the sample pad, gold binding pad, reaction membrane, and absorption pad are arranged in sequence along the flow direction of the sample, and cut the test strips and then put them into the card shell to obtain the eclampsia double test detection card.

[0079] Example 2 Use of the eclampsia dual test card (fluorescence immunochromatography)

[0080] The test card prepared in Example 1 was used for quantitative detection of PlGF and sFlt-1 to assist in predicting the risk of preeclampsia in pregnant women. Specifically, 120 μL of mixed antigens of PlGF and sFlt-1 of different concentrations (0, 5 pg / mL, 25 pg / mL, 100 pg / mL, 250 pg / mL, 500 pg / mL, 1000 pg / mL) were taken and added dropwise to the sample pad of the test card prepared in Example 1. After 15 minutes of sample addition, the test card was inserted into a dry fluorescent immunoassay analyzer (Lanbo AFS-1000) for detection.

[0081] The T / C values ​​of the two indicators obtained with different antigen concentrations are shown in Table 1.

[0082] Table 1

[0083]

[0084]

[0085] Among them, T / C means the ratio of the absolute values ​​of the fluorescence intensity of the test line T and the quality control line C. The larger the value of T / C, the higher the intensity of the test line T and the higher the concentration of the antigen to be tested in the sample. S / N is the ratio of the T / C of the latter concentration to the T / C of the previous concentration. The higher the value, the greater the discrimination between the two concentrations.

[0086] As shown in Table 1, in the joint detection of PlGF and sFlt-1, several combinations of particle sizes (100+100, 200+200, 300+300, 200+300 and 300+100) could not distinguish low concentrations of PlGF; the remaining combinations were 100+200 and 100+300. In contrast, the combination of 100+300 had good distinction between low concentrations of PlGF and sFlt-1. The T / C after changing the particle size and the discrimination at low concentration (i.e., S / N, which is the ratio of the T / C of the latter concentration to the T / C of the previous concentration, the higher the value, the greater the discrimination between the two concentrations) were improved compared with those before changing the particle size. It can be seen that the detection effect of using smaller particle size fluorescent microspheres to label PlGF and larger particle size fluorescent microspheres to label sFlt-1 is the best, which can effectively reduce the mutual interference between different indicators and improve the sensitivity of the two indicators.

[0087] Example 3 Preparation of Pepsinogen I / Pepsinogen II (PGI / PGII) Combined Detection Card (Fluorescence Immunochromatography)

[0088] (1) Fluorescent microspheres of different sizes labeled with mouse anti-PGI and mouse anti-PGII monoclonal antibodies

[0089] Measure 0.09 mg of mouse anti-PGI monoclonal antibody for labeling, dilute to 0.18 ml with purified water and mix well. Then add 1.08 ml of purified water and 0.075 ml of 500 mM MES solution, mix well, add 0.15 ml of fluorescent microspheres with a particle size of 200 nm, and mix with a mixer at room temperature and away from light for 15 minutes. Then add 0.015 ml of 10 mg / mL EDC, mix well immediately, and mix with a mixer at room temperature and away from light for 60 minutes. Centrifuge at 12000-13000 rpm and 4°C for 10 minutes. Carefully discard the supernatant, add 1.5 ml of 25 mM MES, and sonicate for 2 seconds * 10 times to re-dissolve (if the precipitate is difficult to re-dissolve, increase the number of sonications until it is completely re-dissolved). Repeat the centrifugation and 25mM MES washing and re-dissolving process once, then centrifuge for the third time at 4℃ for 10 minutes, carefully discard the supernatant, add 1.5ml fluorescent resuspension solution, and sonicate for 2 seconds*10 times to re-dissolve (if the precipitate is difficult to re-dissolve, increase the number of sonications until it is completely re-dissolved), and obtain the PGI antibody labeled with 200nm fluorescent microspheres. Store at 2-8℃ for future use, with a shelf life of 1 month.

[0090] The same method was used to prepare PGII antibody labeled with 200nm fluorescent microspheres, PGI antibody labeled with 300nm fluorescent microspheres, and PGII antibody labeled with 100nm fluorescent microspheres.

[0091] (2) Preparation of fluorescent microsphere binding pad

[0092] Before each use, the fluorescent microsphere-labeled PGI antibody and fluorescent microsphere-labeled PGII antibody solutions need to be balanced to room temperature and then ultrasonically dissolved for 2 seconds * 10 times. When mixing, surface area correction is performed, that is, both indicators are labeled with 200nm particle size microspheres, and they are diluted 20 times with fluorescent resuspension solution before use (the volume ratio of the two fluorescent labeled antibody solutions to the fluorescent resuspension solution is 0.5:0.5:9). Spray the diluted fluorescent microsphere-labeled antibody on the glass fiber membrane at a spraying volume of 2.5μL / cm 2 After spraying, place it in a drying room to dry for 3-24 hours to obtain the fluorescent conjugate pad, and then store it in a dryer at room temperature. When the particle size is reduced to 100nm, the amount of fluorescent labeled antibody added is 4 times that of 200nm; when the particle size is increased to 300nm, the amount of fluorescent labeled antibody added is 0.5 times that of 200nm.

[0093] The preparation of the fluorescent resuspension is as follows:

[0094] Weigh 6.06 g of tris(hydroxymethyl)aminomethane, 20.00 g of sucrose, 5.00 g of bovine serum albumin, 1.00 g of PEG20000, 1.00 g of Triton X-100, and 1.0 mL of PC300, add 700.0 mL of purified water, stir to fully dissolve, add appropriate amount of concentrated hydrochloric acid, stir evenly, adjust the pH to 7.4, stir to dissolve, and dilute to 1000.0 mL.

[0095] (3) Preparing a test line and a quality control line on a nitrocellulose membrane to obtain a reaction membrane.

[0096] Take 10.0 mg of mouse anti-PGI monoclonal antibody for coating, add it to 3.0 mL 0.05 M PBS, mix thoroughly and dilute to a final volume of 5.0 mL with 0.05 M PBS buffer, the final concentration of the antibody is 2.0 mg / mL. Take 10.0 mg of mouse anti-PGII monoclonal antibody for coating, add it to 3.0 mL 0.05 M PBS, mix thoroughly and dilute to a final volume of 5.0 mL with 0.05 M PBS buffer, the final concentration of the antibody is 2.0 mg / mL. Take 10.0 mg of goat anti-mouse IgG polyclonal antibody (from Luoyang Baitai Biotechnology Co., Ltd.), add it to 3.0 mL 0.05 M PBS buffer, mix thoroughly and dilute to a final volume of 5.0 mL with 0.05 M PBS buffer, the final concentration of the goat anti-mouse IgG polyclonal antibody is 2.0 mg / mL.

[0097] The solution containing mouse anti-PGI, PGII monoclonal antibodies and goat anti-mouse IgG for coating was sprayed at a rate of 10 μL / cm 2 Spray onto the nitrocellulose membrane to form 2 test lines and 1 quality control line, which are test line T1 (PGI), test line T2 (PGII) and quality control line C (sheep anti-mouse IgG) along the sample chromatography direction (i.e. from the conjugate pad to the absorbent paper). Put the reaction membrane coated with the test line and quality control line into the drying workshop for 3-72 hours.

[0098] (4) Paste the sample pad, fluorescent microsphere conjugate pad, reaction membrane, and absorption pad on a PVC plate so that the sample pad, gold conjugate pad, reaction membrane, and absorption pad are arranged in sequence along the flow direction of the sample, and cut the test strips and then load them into the card shell to obtain a pepsinogen I / pepsinogen II (PGI / PGII) combined detection card.

[0099] Example 4 Use of Pepsinogen I / Pepsinogen II (PGI / PGII) Combined Detection Card (Fluorescence Immunochromatography)

[0100] The test card prepared in Example 3 was used for quantitative detection of PGI and PGII, for evaluating the function of gastric oxyntic gland cells, and for assisting in the diagnosis of gastric fundic mucous gland lesions. Specifically, 120 μL of mixed antigens of PGI and PGII of different concentrations (0, 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL) were taken and added dropwise to the sample pad of the test card prepared in Example 3. After 15 minutes of adding the sample, the test card was inserted into a dry fluorescent immunoassay analyzer (Lanbo AFS-1000) for detection.

[0101] The T / C values ​​of the two indicators obtained with different antigen concentrations are shown in Table 2.

[0102] Table 2

[0103]

[0104]

[0105] The T / C of the above two indicators was plotted against the concentration, and the x and y axes were log-converted and then linearly fitted to obtain Table 3. Among them, r is the correlation coefficient of the linear fitting after the T / C was plotted against the concentration, and the closer r is to 1, the higher the correlation.

[0106] Table 3

[0107]

[0108] As can be seen from Tables 2 and 3, in the joint detection of PGI and PGII, the correlation coefficient r of the straight line fitting after the T / C of the same particle size combination (200+200) is plotted against the concentration is relatively low (less than 0.95), indicating that there is a strong mutual interference between the two indicators. In contrast, the combination of 100+300 has a better distinction for low-concentration PGII. The T / C after changing the particle size and the S / N at low concentration are both improved compared to before changing the particle size; and the correlation coefficient r of the two indicators of PGI and PGII has been greatly improved, indicating that the interference between the two indicators has been effectively reduced. It can be seen that the detection effect of using smaller-sized fluorescent microspheres to label PGII and larger-sized fluorescent microspheres to label PGI is better, which can effectively reduce the mutual interference between different indicators and improve the sensitivity of the indicators.

Claims

1. A multi-index test strip, which comprises a sample pad, a binding pad, a reaction membrane and an absorption pad in sequence along the sample flow direction; The conjugate pad comprises an antibody labeled with a first fluorescent microsphere and an antibody labeled with a second fluorescent microsphere, wherein the antibody labeled with the first fluorescent microsphere binds to a first antigen, and the antibody labeled with the second fluorescent microsphere binds to a second antigen; A detection line and a quality control line are sequentially arranged on the reaction membrane along the sample flow direction, wherein the detection line comprises a first capture antibody paired with the antibody labeled with the first fluorescent microsphere, and a second capture antibody paired with the antibody labeled with the second fluorescent microsphere; The first antigen in the sample to be tested is more difficult to detect than the second antigen, and the particle size of the first fluorescent microsphere is smaller than the particle size of the second fluorescent microsphere.

2. The multi-indicator test strip according to claim 1, wherein the first capture antibody is closer to the absorption pad than the second capture antibody, and preferably, the distance between the first capture antibody and the second capture antibody is 1 mm to 10 mm.

3. The multi-index test strip according to claim 1 or 2, wherein the particle size of the first fluorescent microsphere is 50nm to 400nm, and preferably, the particle size of the second fluorescent microsphere is 200nm to 1000nm.

4. The multi-index test strip according to any one of claims 1 to 3, wherein the particle size of the second fluorescent microsphere is greater than that of the first fluorescent microsphere by more than 50 nm, preferably more than 100 nm.

5. The multi-indicator test strip according to any one of claims 1 to 4, wherein the first antigen in the test sample is more difficult to detect than the second antigen, comprising: The first antigen has a lower concentration than the second antigen; The first antigen has a lower detection sensitivity than the second antigen; The first antigen is more susceptible to interference than the second antigen.

6. The multi-index test strip according to any one of claims 1 to 5, wherein the first antigen is placental growth factor (PlGF), and the second antigen is soluble fms-like tyrosine kinase-1 (sFlt-1).

7. The multi-index test strip according to any one of claims 1 to 6, wherein the first antigen is pepsinogen II (PGII) and the second antigen is pepsinogen I (PGI).

8. A multi-index detection card, comprising a card housing and the multi-index test strip according to any one of claims 1 to 7 in the card housing.

9. Use of the multi-index test strip according to any one of claims 1 to 7 in preparing a product for detecting eclampsia.

10. Use of the multi-index test strip according to any one of claims 1 to 7 in preparing a product for detecting gastric function.

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